Summary
Riboswitches are RNA elements capable of modulating gene expression through interaction with cellular metabolites. One member of the riboswitch family, the glmS riboswitch, is unique among riboswitches in that it modulates gene expression by undergoing self-cleavage in the presence of its metabolite glucosamine-6-phosphate (GlcN6P). In order to investigate the interactions between the glmS RNA and GlcN6P we performed nucleotide analog interference mapping (NAIM) and suppression (NAIS). These techniques have been previously used to identify important functional groups in and tertiary contacts necessary for self-splicing and self-cleaving by catalytic RNAs, RNA–protein complexes, RNA folding, and RNA–metal ion interactions. Described here are the details of NAIM and NAIS experiments we have utilized to investigate RNA–ligand interactions between the glmS riboswitch and GlcN6P. These techniques can be employed to study a wide variety of RNA–small molecule interactions.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Strobel, S. and Shetty, K. (1997). Defining the chemical groups essential for Tetrahymena group I intron function by nucleotide analog interference mapping. Proc. Natl. Acad. Sci. U. S. A. 94, 2903–2908.
Ryder, S. and Strobel, S. (1999). Nucleotide analog interference mapping. Methods 18, 38–50.
Ryder, S., Ortoleva-Donnelly, L., Kosek, A. and Strobel, S. (2000). Chemical probing of RNA by nucleotide analog interference mapping. Methods Enzymol. 317, 92–109.
Gish, G. and Eckstein, F. (1988). DNA and RNA sequence determination based on phosphorothioate chemistry. Science 240, 1520–1522.
Strobel, S., Ortoleva-Donnelly, L., Ryder, S., Cate, J. and Moncoeur, E. (1998). Complementary sets of noncanonical base pairs mediate RNA helix packing in the group I intron active site. Nat. Struct. Biol. 5, 60–66.
Szewczak, A., Ortoleva-Donnelly, L., Ryder, S., Moncouer, E. and Strobel, S. (1998). A minor groove triple helix in the active site of the Tetrahymena group I intron. Nat. Struct. Biol. 5, 1037–1042.
Soukup, J., Minakawa, N., Matsuda, A. and Strobel, S. (2002). Identification of A-minor tertiary interactions within a bacterial group I intron active site by 3-deazaadenosine interference mapping. Biochemistry 41, 10426–10438.
Jansen, J.A., McCarthy, T.J., Soukup, G.A. and Soukup, J.K. (2006). Backbone and nucleobase contacts to glucosamine-6-phosphate in the glmS ribozyme. Nat. Struct. Mol. Biol. 13, 517–523.
Winkler, W.C., Nahvi, A., Roth, A., Collins, J.A. and Breaker, R.R. (2004) Control of gene expression by a natural metabolite-responsive ribozyme. Nature 428, 281–286.
McCarthy, T.J., Plog, M.A., Floy, S.A., Jansen, J.A., Soukup, J.K. and Soukup, G.A. (2005). Ligand requirements for glmS ribozyme self-cleavage. Chem. Biol. 12, 1221–1226.
Christian, E. and Yarus, M. (1993). Metal coordination sites that contribute to structure and catalysis in the group I intron from Tetrahymena. Biochemistry 32, 4475–4480.
Chanfreau, G. and Jacquier, A. (1994). Catalytic site components common to both splicing steps of a group II intron. Science 266, 1383–1387.
Hardt, W., Warnecke, J., Erdmann, V. and Hartmann, R. (1995). Rp-phosphorothioate modifications in RNase P RNA that interfere with tRNA binding. EMBO J. 14, 2935–2944.
Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. (Nolan, C., ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Ortoleva-Donnelly, L., Szewczak, A.A., Gutell, R.R. and Strobel, S. (1998). The chemical basis of adenosine conservation throughout the Tetrahymena ribozyme. RNA 4, 498–519.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Soukup, J.K., Soukup, G.A. (2009). Identification of Metabolite–Riboswitch Interactions Using Nucleotide Analog Interference Mapping and Suppression . In: Serganov, A. (eds) Riboswitches. Methods in Molecular Biology, vol 540. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-558-9_14
Download citation
DOI: https://doi.org/10.1007/978-1-59745-558-9_14
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-934115-88-6
Online ISBN: 978-1-59745-558-9
eBook Packages: Springer Protocols